The fungal pathogen Cryptococcus neoformans causes a high incidence of life-threatening infections in AIDS patients and this fungus therefore poses a major threat to the estimated 40 million people worldwide who are infected with HIV. In this context, our long-term objectives are to develop a detailed understanding of virulence in C. neoformans through a combination of genomic and molecular genetic approaches and to contribute the knowledge that we acquire to strategies to combat fungal infections. Our first specific aim is to determine the gene content in the two capsular serotypes of C. neoformans (A and D) that represent the global population of isolates that infect AIDS patients. To achieve this goal, we will perform comparative DNA hybridization experiments with whole genome arrays of bacterial artificial chromosome (BAC) clones. The arrays will be developed from physical maps of BAC clones generated by a fingerprinting method; maps have already been constructed with this method for three isolates of C. neoformans. Comparative hybridization will identify regions of difference between strains and these regions will be sequenced and compared to existing genomic sequence information to develop a view of variability in gene content within and between serotypes. We will also examine the conservation of a gene cluster with a putative role in iron acquisition. The second specific aim is to characterize the transcriptomes of two strains of C. neoformans representing the A and D serotypes during pulmonary infection in a murine model and after phagocytosis by a macrophage-like cell line. We will use serial analysis of gene expression (SAGE) to identify the relative abundance of transcripts on a genomic scale. The new data that we obtain will be compared between the serotypes and compared with SAGE data that we have already collected from C. neoformans cells that are responding to different temperatures (25 degrees C vs. 37 degrees C) and iron levels. The third specific aim is to perform a molecular genetic analysis of the cluster of iron-regulated genes that was identified by our SAGE analysis in a serotype D strain. These genes encode a putative multicopper oxidase (CnFET3) and a putative iron permease (CnFTR1). We will compare the organization and transcriptional regulation of these genes (and a third iron-regulated gene CnURF1) between serotypes. We will also disrupt the genes to examine their role in virulence.